Covalent Modification of Biomolecules through Maleimide-Based Labeling Strategies

Abstract

Since their first use in bioconjugation more than 50 years ago, maleimides have become privileged chemical partners for the site-selective modification of proteins via thio-Michael addition of biothiols and, to a lesser extent, via Diels-Alder (DA) reactions with biocompatible dienes. Prominent examples include immunotoxins and marketed maleimide-based antibody-drug conjugates (ADCs) such as Adcetris, which are used in cancer therapies. Among the key factors in the success of these groups is the availability of several maleimides that can be N-functionalized by fluorophores, affinity tags, spin labels, and pharmacophores, as well as their unique reactivities in terms of selectivity and kinetics. However, maleimide conjugate reactions have long been considered irreversible, and only recently have systematic studies regarding their reversibility and stability toward hydrolysis been reported. This review provides an overview of the diverse applications for maleimides in bioconjugation, highlighting their strengths and weaknesses, which are being overcome by recent strategies. Finally, the fluorescence quenching ability of maleimides was leveraged for the preparation of fluorogenic probes, which are mainly used for the specific detection of thiol analytes. A summary of the reported structures, their photophysical features, and their relative efficiencies is discussed in the last part of the review.

Detection of Biothiols with a Fast‐Responsive and Water‐Soluble Pyrazolone‐Based Fluorogenic Probe

“…A number of reported probes lose their fluorogenic properties under weakly acidic environments [34][35][36][37] due to the predominance of the thiol form over thiolate anion. [38] It is, however, of interest to have probes that are fluorescent over the entire biologically relevant pH range from 4 to 8. This is particularly true for the fluorescence labelling of endocytic compartments, which are intracellular acidic organelles whose internal pH is around 5-6.…”

A Green BODIPY‐Based, Super‐Fluorogenic, Protein‐Specific Labelling Agent

“…Thea ddition of at hiol to am aleimide is chemoselective, fast, high yielding and results in the formation of as table thioether product. [28,25] When applied to protein labelling,the robust covalent bonds formed with the target protein should be stable even if the labelled protein is then denatured. To test this hypothesis,w el abelled MBP-dC10a with YC23 in bacterial lysate and heated the sample to 95 8 8Cf or 15 min, prior to analysis by SDS-PAGE(see Supporting Information).…”

“…FlARe reagents comprise af luorophore and two maleimide groups.W hile the chemoselective thiol addition reaction of maleimides has been broadly applied to bioconjugation, [28] maleimides can also quench fluorescence by photoinduced electron transfer (PeT). [14] We have shown [23,24] that the latent fluorescence of our fluorogens is restored only after both maleimide groups have undergone addition reactions (Scheme 1).…”

Design and synthesis of cysteine-specific labels for photo-crosslinking studies

“…N-Maleimido-diazirine 3 can be used to label thiols on peptides or proteins via a conventional conjugate addition, used widely in protein labelling. 19 MTS-alkynyldiazirine 4 can be used to label thiols on peptides or proteins, yet bears an additional bio-orthogonal group (the alkyne) that could be exploited to introduce further functionality (e.g. uorophore, biotin) by "click" chemistry 20,21 to support chemical proteomics workows.…”